Tumor suppressor p53 protein binds to DNA and represses transcriptional activation at the G1-S boundary. Since repressor activity requires translocation of p53 to the nucleus, we are interested in the biochemical events involved in synthesis and regulation of the p53 protein complex. We have shown a number of proteins are bound heat shock proteins (HSP) complexed to p53 while in the cytoplasm but released when the complex enters the nucleus, and our lab has shown there is a family of at least 11 p53 isoforms which originate from the same locus but are post- translationally modified. We have also determined there is a conformational change in both p53 and HSP's once they enter the nucleus. Since the reasons for complex formation and the biochemical function of the associated proteins which bind to p53 is largely unknown, we plan to isolate roles in translocation. We have obtained a series of truncated human and rodent p53 constructs missing various segments of the gene including the transactivation, binding and oligomerization domains. The rodent gene is missing the nuclear localization signal. We have prepared nine constructs of the rodent genes into mammalian expression vectors containing a neo selection marker and then successfully transfected them into murine (10)1 cells, which are p53 null. We plan to develop the human constructs this year. Since the human genes are missing start and stop codons in some cases we will need to prepare special primers to replace these codons. These modified genes should allow ample formation of p53 without it translocating it to the nucleus and enable isolation of the p53 complex and its associated proteins. These constructs will be used to probe the respective roles of p53-complexed proteins in formation and stability of the p53 structure, as well as the mechanism of complex translocation to the nucleus and release of p53 for attachment to DNA.